Cryo-EM structures of the human NaS1 and NaDC1 transporters revealed the elevator transport and allosteric regulation mechanism

The solute carrier 13 (SLC13) family comprises electrogenic sodium ion–coupled anion cotransporters, segregating into sodium ion–sulfate cotransporters (NaSs) and sodium ion–di- and–tricarboxylate cotransporters (NaDCs). NaS1 and NaDC1 regulate sulfate homeostasis and oxidative metabolism, respectively. NaS1 deficiency affects murine growth and fertility, while NaDC1 affects urinary citrate and calcium nephrolithiasis. Despite their importance, the mechanisms of substrate recognition and transport remain insufficiently characterized. In this study, we determined the cryo–electron microscopy structures of human NaS1, capturing inward-facing and combined inward-facing/outward-facing conformations within a dimer both in apo and sulfate-bound states. In addition, we elucidated NaDC1’s outward-facing conformation, encompassing apo, citrate-bound, and N-(p-amylcinnamoyl) anthranilic acid (ACA) inhibitor–bound states. Structural scrutiny illuminates a detailed elevator mechanism driving conformational changes. Notably, the ACA inhibitor unexpectedly binds primarily anchored by transmembrane 2 (TM2), Loop 10, TM11, and TM6a proximate to the cytosolic membrane. Our findings provide crucial insights into SLC13 transport mechanisms, paving the way for future drug design.


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Figs. S1 to S14 Tables S1 and S2     Please refer to the Materials and Methods, Fig. S5 and Table S2 for details.

Fig. S3 .
Fig. S3.Cryo-EM analysis of the NaS1 apo state.(A) Local resolution map and Euler angle distribution for the 3D reconstruction of NaS1 apo state (NaS1apo-IN/IN) are shown in the upper two panels.The left lower panel is the FSC curve of the refined model of NaS1 versus the cryo-EM map that it is refined against (black); of the model refined against the first half map versus the same map (red); and of the model refined against the first half map versus the second half map (green).The small difference between the red and green curves indicates that the refinement of the atomic coordinates did not suffer from overfitting.The right lower panel is the Gold standard FSC curve for the 3D refinement of the NaS1apo-IN/IN state.(B) same as a but for NaS1 apo state (NaS1apo-IN/OUT).

Fig. S5 .
Fig. S5.Cryo-EM analysis of the NaS1 in complex with sulfate.(A) Local resolution map and Euler angle distribution for the 3D reconstruction of NaS1 binding to sulfate (NaS1sulfate-IN/IN) are shown in the upper two panels.The left lower panel is the FSC curve of the refined model of NaS1 versus the cryo-EM map that it is refined against (black); of the model refined against the first half map versus the same map (red); and of the model refined against the first half map versus the second half map (green).The small difference between the red and green curves indicates that the refinement of the atomic coordinates did not suffer from overfitting.The right lower panel is the Gold standard FSC curve for the 3D refinement of the NaS1sulfate-IN/IN state.(B) same as a but for NaS1 binding to sulfate (NaS1sulfate-IN/OUT).

Fig. S6 .
Fig. S6.Flowchart for cryo-EM data processing of NaS1 complex with sulfate structures.Please refer to the Materials and Methods, Fig. S7 and TableS2for details.

Fig. S7 .
Fig. S7.Cryo-EM analysis of the NaDC1.(A) Representative cryo-EM micrograph as well as 2D class averages results.The scale bar in 2D class averages is 10 nm.(B) Gold standard FSC curve for the 3D refinement of the NaDC1 apo state (blue), NaDC1 in complex with ACA (orange), and NaDC1 in complex with citrate (green), respectively.(C) Local resolution map and Euler angle distribution for the 3D reconstruction of NaDC1 in complex with ACA (NaDC1_ACA) are shown in the left and middle.FSC curve of the refined model of NaDC1_ACA versus the cryo-EM map that it is refined against (black); of the model refined against the first half map versus

Fig. S8 .
Fig. S8.Flowchart for cryo-EM data processing of NaDC1 structures.Please refer to the Materials and Methods, Fig. S3 and TableS1for details.

Fig. S9 .
Fig. S9.Cartoon of overall structures of NaS1 and NaDC1.(A) Cartoon of overall structures of NaS1 and NaDC1.Only one subunit in each protein is colored in rainbow.(B) cryo-EM map of NaS1_IN/OUT.The OUT subunit is colored according to domains.

Fig. S10 .
Fig. S10.Confocal images of HEK-293T cells expressing NaDC1 or mutants.(A) Representative traces of currents of WT-NaDC1 and I550V by 10 mM citrate.(B) Quantification of currents of WT-NaDC1 and I550V.ns, no significance, calculated by t-test.(C) Representative images of GFP signaling of NaDC1 and mutants transfected HEK-293T cells.Scale bar = 20 µm.(D) Quantification of fluorescence intensity.Note the GFP signal is absent in blank HEK293T cells.ns, no significance, calculated by One-way ANOVA.All data present Mean ± SEM.

Fig. S11 .
Fig. S11.Substrate binding pockets are highly conserved in SLC13 proteins.(A) Sulfate binding pocket in NaS1_apo_IN/IN.The sodium ions are bound to Na1 and Na2 sites, of which the cryo-EM density map are shown in mesh.(B) Sulfate binding in outward facing protomer of NaS1_sulfate_IN/OUT. Sodium density near L10 is not obvious in cryo-EM map.(C) Sulfate binding in inward facing protomer of NaS1_sulfate_IN/OUT.Both sodium densities are not obvious in cryo-EM map.(D) Sulfate binding of different subunits are alike in NaS1_IN/OUT.(E) Although the density of citrate is not continuous in cryo-EM map probably due to conformation flexibility, there is no obvious density around citrate binding pocket in NaDC1_apo

Fig. S14 .
Fig. S14.Conformational difference between SLC13s.(A) Left: conformational comparison between NaDC1 and LalNDY (PDB ID: 6WU1).The structure of LalNDY is reported to be in the outward conformation.When the scaffold domains from NaDC1 and LalNDY is aligned, the core domain of NaDC1 moves further torwards the extracellular space, indicating a new outward-open conformation.Middle, conformational comparison between NaDC1 and NaS1.The structures are aligned to the scaffold domain, the core domain of NaDC1 moves torwards the extracellular space.Right, conformational comparison between NaS1 and NaCT (PDB ID: 7JSK).When